Crystalline cell surface layer of Mycobacterium bovis BCG.

A paracrystalline surface layer (S layer) was found as the outermost layer of the cell wall of five Mycobacterium bovis BCG strains. An oblique arrangement of the subunits in the S layer was only clearly seen in thin-sectioned and shadowed preparations, and the unit constant was about 5.5 nm.     

Cultivation of Mycobacterium bovis BCG in bioreactors

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.     

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.     

Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 × 10³ to 3.6 × 10³ gene copies g of soil⁻¹, depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37°C with moist soil (−20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Characterization of PPD protein antigens in whole cell lysates of Mycobacterium bovis BCG

The low molecular mass protein antigens in PPD from M. bovis BCG were chemically oligomerized using sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (S-SMPB) as a crosslinking agent. Protein oligomers with molecular mass over 90 kDa were obtained and used for the preparation of hyperimmune polyclonal rabbit antiserum. Using this antiserum four protein bands with molecular mass 120, 90, 75 and 65 kDA were detected in immunoblotting analysis of sonic extract from M. bovis BCG separated in SDS-polyacrylamide gel. We suggest that these immunoreactive proteins in the sonic extract represent the native forms of the heat stable low molecular mass protein antigens in PPD.     

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.     

The uraA locus and homologous recombination in Mycobacterium bovis BCG.

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity. The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines.     

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.     

Structural definition of arabinomannans from Mycobacterium bovis BCG

The structures of the hydrophilic parietal and cellular arabinomannans isolated from Mycobacterium bovis BCG cell wall [Nigou et al. (1997) J Biol Chem 272: 23094-103] were investigated. Their molecular mass as determined by MALDI-TOF mass spectrometry was around 16 kDa. Concerning cap structure, capillary electrophoresis analysis demonstrated that dimannoside (Manpalpha1-->2Manp) was the most abundant motif (65-75%). Using two-dimensional 1H-13C NMR spectroscopy, the mannan core was unambiguously demonstrated to be composed of -->6Manpalpha1--> backbone substituted at some O-2 by a single Manp unit. The branching degree was determined as 84%. Finally, arabinomannans were found to be devoid of the phosphatidyl-myo-inositol anchor and, by aminonaphthalene disulfonate tagging, the mannan core was shown to contain a reducing end. This constitutes the main difference between arabinomannans and lipoarabinomannans from Mycobacterium bovis BCG.     

The role of histamine in the intracellular survival of Mycobacterium bovis BCG

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.     

DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.     

Gene replacement by homologous recombination in Mycobacterium bovis BCG

Gene replacement by homologous recombination is a powerful tool for fundamental studies of gene function, as well as allowing specific attenuation of pathogens, but has proved difficult to achieve for Mycobacterium tuberculosis. We have used a plasmid-based test system to demonstrate the occurrence of homologous recombination in the tuberculosis vaccine strain Mycobacterium bovis BCG, and we have successfully replaced a target gene in BCG by homologous recombination, using a shuttle plasmid. Specific inactivation of selected genes will facilitate study of virulence factors and drug resistance as well as allowing rational attenuation of M. tuberculosis for the production of new vaccines.     

Functional expression of the Flp recombinase in Mycobacterium bovis BCG

Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants because there are only two efficient antibiotic resistance genes available for allelic exchange experiments. Sequence-specific recombinbases such as the Flp recombinase can be used to excise resistance markers. Expression of the flp(e) gene from Saccharomyces cerevisiae is functional for this purpose in fast-growing Mycobacterium smegmatis but not in slow-growing mycobacteria such as M. bovis BCG or M. tuberculosis. We synthesized the flp(m) gene by adapting the codon usage to that preferred by M. tuberculosis. This increased the G+C content from 38% to 61%. Using the synthetic flp(m) gene, the frequency of removal of FRT-hyg-FRT cassette from the chromosome by the Flp recombinase was increased by more than 100-fold in M. smegmatis. In addition, 40% of all clones of M. bovis BCG had lost the hyg resistance cassette after transient expression of the flp(m) gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flp(m) encoded Flp recombinase is not only an improved genetic tool for M. smegmatis, but can also be used in slow growing mycobacteria such as M. tuberculosis for constructing unmarked mutations. Other more sophisticated applications in mycobacterial genetics would also profit from the improved Flp/FRT system.     

Survival of Mycobacterium tuberculosis and Mycobacterium bovis BCG in lysosomes in vivo

Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro, was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis.     

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

Whole-genome sequences of four Mycobacterium bovis BCG vaccine strains

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only vaccine available against tuberculosis (TB). A number of BCG strains are in use, and they exhibit biochemical and genetic differences. We report the genome sequences of four BCG strains representing different lineages, which will help to design more effective TB vaccines.     

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.